Actin filament lengths are precisely regulated and very stable in the erythrocyte membrane skeleton and the sarcomers of striated muscle. In motile cells, actin filament lengths are dynamically regulated when cells crawl or change shape, extending lamellipodia and filopodia. The broad, long term objective of this research is to elucidate how actin dynamics at filament ends maintains stable filament lengths in cells such as erythrocytes and muscle, yet also controls actin turnover in labile filament networks in motile cells. An additional long-term objective is to determine how actin dynamics at filament ends influences cell shape and deformability of normal and abnormal erythrocytes. This proposal focuses on the role of tropomodulin, a tropomyosin binding and actin pointed end capping protein. The specific aims are 1) to characterize tmod actin capping and muscle and nonmuscle tm binding domains in vitro and to identify new tmod binding partner in erythrocytes, using biochemical and biophysical approaches. 2) To determine the mechanistic relationship between tmod targeting to pointed ends, actin dynamics and regulation of filament lengths in living cardiac muscle cells, using photo bleaching of GFP-tmods, microinjection of rho-actin, phalloidin staining and deconvolution analysis. 3) to test the hypothesis that actin filament ends are dynamic in the erythrocyte membrane skeleton, rhodamine or biotin labeled actins will be introduced into resealed ghosts and subunit exchange or polymerization evaluated by light and electron microscopy and quantitative binding assays. Actin perturbing drugs and ektacytometry will be used to investigate a role for actin dynamics in membrane deformability and stability of intact cells and resealed ghosts. Actin dynamics in abnormal erythrocytes from beta and alpha adducin knockout mice will be used to test a role for adducin in regulating actin dynamics at barbed ends. 4) We will characterize the expression, localization and function of a new Tmod isoform, U-Tmod, in epithelial cells in tissues and in the leading lamellipodia of migrating epithelial cell in culture. The role of Tmod domains will be investigated by evaluating abilities of GFP-U-Tmod and Tmod fragment to target to the leading lamellipodia, and we will test the hypothesis that tmod plays a role in cell motility by overexpressing GFP-U-TMod in migrating cells, followed by evaluation of rho actin dynamics and leading edge extension in living cells.